PRODUCTION AND CHARACTERIZATION OF CHITINASE ENZYMES FROM SULILI HOT SPRING IN SOUTH SULAWESI, Bacillus sp. HSA,3-1a | Natsir | Indonesian Journal of Chemistry 21470 40556 1 PB

Indo. J. Chem., 2010, 10 (2), 263 - 267

263

PRODUCTION AND CHARACTERIZATION OF CHITINASE ENZYMES FROM SULILI HOT
SPRING IN SOUTH SULAWESI, Bacillus sp. HSA,3-1a
Hasnah Natsir1.*, Abd. Rauf Patong1, Maggy Thenawidjaja Suhartono2, and Ahyar Ahmad1
1

2

Department of Chemistry, Faculty of Mathematics and Natural Sciences, Hasanuddin University,
Jl. Perintis Kemerdekaan Km 10 Tamalanrea, Makassar 90245, South Sulawesi, Indonesia

Departement of Food Technology and Human Nutrition FATETA and Research Center for Biotechnology, Bogor Agricultural
University, Jl. Raya Darmaga, Campus IPB, PO.BOX 220, Bogor 16002, West Java, Indonesia
Received March 2, 2010; Accepted June 2, 2010

ABSTRACT
Chitinase is an extracellular enzyme which is capable in hydrolyzing insoluble chitin to its oligomeric and
monomeric components. The enzyme produced by thermophilic bacteria was screened and isolated from Sulili hot

spring in Pinrang, South Sulawesi, Indonesia. The gram positive spore forming rod shape bacteria was identified as
Bacillus sp. HSA,3-1a through morphological and physiological analysis. The production of chitinase enzyme was
conducted at various concentration of colloidal chitin at a pH of 7.0 and a temperature of 55 °C. The bacteria
optimally was produced the enzyme at a colloidal chitin concentration of 0.5% after 72 h of incubation. The optimum
temperature, pH and substrate concentration of chitinase were 60 °C, 7.0 and 0.3%, respectively. The enzyme was
stable at a pH of 7.0 and a temperature of 60 °C after 2 h of incubation. The chitinase activities was increased by
2+
2+
2+
2+
2+
2
addition of 1 mM Mg , Ca and Mn ions, whereas the activities were decreased by 1 mM Co , Fe and Zn ions.
The molecular weight of the crude enzyme was determined using SDS-PAGE analysis. Five protein fractions were
obtained from SDS-PAGE, with MWs of 79, 71, 48, 43 and 22 kDa.
Keywords: colloidal chitin, thermophilic bacteria, chitinase
INTRODUCTION
Chitinase (EC 3.2.1.14) is an enzyme which can
hydrolyze chitin to its oligomeric, dimeric and monomeric
component. The enzyme found in numerous bacteria,

fungi, insect, plant and animal involves in natural
protection mechanism. Many bacteria and fungi
containing the chitinolitic enzyme convert chitin into
carbon and nitrogen that can serve as energy source.
Chitinolitic bacteria are typically detected and screened
through the production of clearing zones on chitin
containing agar as selective medium. Chitinases have
many industrial and agricultural applications such as
preparation of chitooligosasccharides, biocontrol of plant
pathogenic fungi, and production of single-cell protein [13].
Chitin is highly insoluble β-(1,4)-linked polymer
composed primarily of N-acetyl-glucosamine and some
glucosamine residues, and is believed to be the second
most abundant biomaterial after cellulose. It is widely
distributed as a structural component of crustaceaes,
insects, marine invertebrates, such as cuttlefish, crab,
and lobster as well as a component of the cell walls fungi
and algae, the annual production in marine water alone
is estimated to be more than 100 billion tons [2,4-5].

* Corresponding author. Tel/Fax : +62-411-586498
Email address : hasnahnatsir@gmail.com

Hasnah Natsir et al.

A research on isolation of microbes producing
chitinase has been successfully conducted. The
researches included acidophilic chitinase isolated from
kamojang Crater of West Java [6] and chitinase from
thermophilic bacteria isolated from hot water source of
Tamposo, North Sulawesi [7].
The previous research on isolation of thermophilic
bacteria from the hot water source in South Sulawesi
produced several isolates of chitinolitic bacteria, but the
optimum condition to produce them and characteristic
of biochemical properties of the isolates have not been
known, yet. Therefore, a further research on the isolate
is necessary. The HSA 3-1a isolate was selected
because the isolate had the highest chitinolitic activity
[8].

In this research, identification of the HAS,3-1a
isolate, production optimalization and characterization
of the crude enzyme were carried out. The research
involved determination of optimum pH and
temperature, pH and temperature stability, the metal
ion which can be function as activator and inhibitor to
the activity of chitinase as well as analysis of molecular
mass of protein with SDS-Page (Sodium Dodecyl
Sulphate Polyacrylamide Gel Electrophoresis).

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Indo. J. Chem., 2010, 10 (2), 263 - 267

EXPERIMENTAL SECTION
Materials
Thermophilic bacteria, HSA-3,1a isolate, colloidal
chitin (chitin sigma, EC. 215-744) as the enzyme
substrate, medium containing yeast extract, bacto
pectone, bacto agar, (NH4)2SO4, K2HPO4, NaCl, MgSO4.

7 H2O, several divalent cations, Bovine Serum Albumin
(BSA) as the protein standard.

Determination of Protein Content
Determination of protein content was conducted
using the Lowry method [10]. Two solutions were used
in the method; Lowry B and Lowry B. The former
consisted of 2% Na2CO3 in a mixture of 0.1 N NaOH,
1% CuSO4, and sodium potassium tartrate with the
ratio of 100:1:1 and the latter was a solution of
phospho-tungstic-phospho-molybdic acid (folin) in
aquadest (1:1). The protein content was measured by a
spectrophotometer at the maximum wavelength using
BSA as the standard.

Instrumentation
Analytical balance (Ohaus), autoclave (Model
8000-DSE Napco), shaker water bath (Memmert),
spectrophotometer, pH meter (Memmet), Centrifuge
(Model 800), hotplate stirrer (Fisher Versamix TM).

Procedure
Microorganism Analysis
The culture stock of thermophilic bacteria, HAS,31a isolate, was cultivated for 18-20 h at the temperature
of 55 °C in the medium of agar slant without colloidal
chitin [6,9]. The morphological identification and the
biochemical test were then conducted. The former
involved bacterial shape, colonies color, cell shape,
gram test (positive or negative) with or without spores,
and mortality test; the latter included reactions: catalase,
oxidase, indol, citrate, gelatin, methyl red and
carbohydrate test [7].
Optimation of Chitinase production [6,9]
Thermophilic bacteria, Bacillus sp., particularly the
HSA-3,1a isolate was inoculated as many as 10%
isolate into the fermentation medium with the same
composition as the medium. The isolate was then
shaken at 180 rpm, 55 °C for 1-6 days. Sampling was
taken every 24 h for analysis of the optical density (OD),
the chitinase activation and the protein content. After
knowing the concentration of colloidal chitin and the

fermentation optimum time, reproduction of chitinase in
the large scale (2L) was conducted for enzyme
characterization.
Characterization of Chitinase [6,9]
The chitinase enzyme produced at the optimum
condition was put in the centrifuge at the speed of 3500
rpm for 30 min and 4 °C. The product was characterized
by: determining optimum temperature and pH,
temperature and pH stability, metal ions functioning as
activator or inhibitor at the concentrations of 1.0 mM and
5.0 mM, with metal ions used of MgCl2; CaCl2; MnCl2;
NiCl2; and CuCl2. Molecular mass analysis of proteins by
a method of SDS-PAGE.

Hasnah Natsir et al.

Determination of Chitinase Activity
The chitinase activity was determined by using
the Ueda and Arai method [11] as follows: 2 mL of
phosphate buffer solution (pH 7) was mixed with 1 mL

of the enzyme solution (sample) and 1 mL of 0.3%
colloidal chitin (substrate). The mixture was incubated
at 55 °C for 60 min. The turbidity caused chitin
remaining in the reaction mixture was measured at the
wavelength of 660 nm.
Definition of one activity unit of chitinase is the
amount of enzyme catalyzing the decrease of
absorbance as many as 0.001 per min per mL enzyme
at the wavelength of 660 nm.
Determination of Chitinase Molecular Mass [10]
A method used to determine the molecular mass
of chitinase was the electrophoresis method using a
LMW (Low Molecular Weight) marker: phosphorylase-b
(Mr: 94,000); BSA (Mr: 67,000); ovalbumin (Mr: 43,000);
carbonic anhydrate (Mr: 30,000); soybean trypsin
inhibitor (Mr: 20,100), and silver stain colorization.
RESULT AND DISCUSSION
It was known from the previous work that the
HSA,3-1a isolate of thermophilic bacteria had the
chitinolitic activity [8]. Morphological and physiological

identification results of the bacteria showed that the
bacteria had properties as follows: they were positive
gram bacteria with the shape of bacillus and with
spores. Based on the identification and Bergey’s
Mannual of Systematic Bacteriology the HSA,3-1a
isolate is in Bacillus genus. (Fig. 1b). Therefore, the
isolate is named Bacillus sp. HSA-3,1a (Fig. 1a).
Results of chitinase production at various
concentrations of colloidal chitin showed that the
enzyme was optimally produced at the colloidal chitin
concentration of 0.5% with the pH medium of 7.0; the
incubation temperature of 55 °C (Fig. 2).
The optimum condition of chitinase production
obtained from the research was similar to the one
obtained from Bacillus MH-1 grown at 58 °C [5] and
from B. licheniformis MB-2 using 0.5% colloidal chitin 0.5%

Indo. J. Chem., 2010, 10 (2), 263 - 267

a

265

b

Fig 1. Bacillus sp. HSA-3,1a: a) the colony of bacteria
grew at 55 °C, pH 7.0 for 3 days. b). cell shape and
spore coloring.

Fig 3. Optimum condition for producing chitinase by the
Bacillus sp. HSA,3-1a at the substrate concentration of
0.5%, the medium pH of 7.0, and the temperature of 55 °C.

Fig 2. The effect of colloidal chitin concentrations on the
extracellular chitinase production from Bacillus sp.
HSA,3-1a.

Fig 4. The chitinase activity of the Bacillus sp. HAS,31a as a function of the incubation temperatures with the
chitin colloid concentration of 0.3% and the phospate
buffer pH of 7.0.

at the cultivation temperature of 50 °C [12]. The optimal
result was used in the chitinase production in order to
find out the optimum time of chitinase production.
The chitinase production achieved the optimum
condition at the end of stationary phase for the growth of
Bacillus sp. HSA,3-1a, i.e. at hour-72 (Fig. 3). This may
be caused by the decrease of nutrition in the medium.
Therefore, the chitinase was secreted to the outside of
cell in the large amount. The chitinase was used to
degrade colloidal chitin acting as the source of carbon
and nitrogen in the bacteria. Thus, the optimum
condition to produce chitinase from Bacillus sp. HSA,31a was as follows: the colloidal chitin concentration of
0.5%; the medium pH of 7.0; temperature of 55 °C and
the fermentation time of 72 h. The condition was used in
producing chitinase for characterization.
Determination of Optimum Temperature and the
Stability
Fig. 4 illustrates the effect of the incubation
temperature on the chitinase activity. It is obvious that
the HAS,3-1a isolate has the high activity in the

Hasnah Natsir et al.

temperature between 55 and 65 °C, but the optimum
temperature is 60 °C (Fig. 4).
The chitinase produced from the isolate is
categorized into a thermophilic enzyme. A stability test
conducted at temperatures of 55 and 60 °C in the preincubation step showed that the enzyme was still stable
for 2 h. This result indicates that the enzyme is
thermostable. A different result was obtained when the
Bacillus licheniformis MB-2 isolate was used. The
chitinase enzyme produced by the isolate was stable
for 3 h at the temperature of 60 °C [12].
Determination of Optimum pH and the Stability
Enzymes can behave as acid, base or neutral
based on their amino acid constitution which can
influence their active sites. The effect of the buffer pH
on the chitinase activity is given in Fig. 5.
It is clear that the chitinase activity increases from
the pH of 6.4 to 7.0 and then decreases at the pH of
7.2. The optimum pH is 7.0. The same optimum pH
was obtained when the enzyme was produced from
Bacillus sp. 13.26 [7]. However, the different optimum
pH was given by the chitinase enzyme isolated from

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Indo. J. Chem., 2010, 10 (2), 263 - 267

Fig 5. The chitinase activity Bacillus sp. HAS, 3-1a
isolate as a function of the phospate buffer pH at the
colloid chitin concentration of 0.3% and the temperature
of 60 °C.

Fig 6. The effect of the substrate concentration on
the chitinase activity from Bacillus sp. HAS, 3-1a at
the temperature of 60oC and the phospate buffer
pH of 7.0.

Fig 7. The Effect of metal ions on chitinase
activities of Bacillus sp. HAS,3-1a at the colloidal
chitin concentration of 0.3%, the temperature of 60 °C
and the phospate buffer pH of 7.0.
different sources; the optimum pH of the enzyme from B.
licheniformis MB-2 was 8.0 [12], and the one from
Bacillus sp. K-22 was 5.0 which are stable for 2 h at the
pre-incubation step [6].

Hasnah Natsir et al.

Effect of Substrate Concentration on Chitinase
Activity
Experiments
with
various
substrate
concentrations were conducted to know the optimum
substrate concentration that can react with the enzyme.
Fig. 6 shows that the chitinase activity for Bacillus sp.
HSA,3-1a increases from the substrate concentrations
of 0.1 to 0.3%.
This indicates that the activity of chitinase
achieves the maximum at the substrate concentration
of 0.3%. The condition is parallel with the chitinase
activity from Bacillus sp. 13.26 and from B.
licheniformis MB-2 which achieve the optimum at the
substrate (colloidal chitin) concentration of 0.3% [7,12].
At the low substrate concentration, the active
sites of enzyme bind only small amount of enzyme, but
the higher the concentration of the substrate the larger
amount of the substrate which can be bound by the
active sites of enzyme. Therefore, the activity of
enzyme will increase with the increase of the substrate
concentration. However, at the certain substrate
concentration, all active sites will be saturated by the
substrate. At this condition, the increase of the
substrate concentration will not increase the enzyme
activity indicating that the substrate concentration has
achieved the optimum condition.
Effect of Cofactor on Enzyme Activity
Generally, the enzyme needs non-protein
compounds, such as, metal ions in doing its catalytic
function. The compounds are required by the enzyme
as components in the active side for activator or
inhibitor in catalyzing the substrate at the certain
concentration.
The pattern of activator or inhibitor effects of
metal ions on the chitinase activity of Bacillus sp.
HSA,3-1a was studied by using divalent ions. Results
2+
showed that chitinase was activated by 1 mM Ca ,
2+
2+
Mg , and Mn ions. The result was similar to the one
given by chitinase of Bacillus MH-1 [5] and of B.
2+
Licheniformis MB-2 [12] activated by 1 mM Ca and
2+
Mg ions. But the chitinase activity was inhibited by 1
2+
2+
2+
mM Co , Zn and Cu ions with competitive inhibition
(Fig. 7).
Activator compounds until the certain amount can
enhance the rate of enzyme catalyzed reaction, but the
excess amount of the activator can cause the
competition of the free activator and the activatorsubstrate complex to enzyme. The excess amount of
the free activator causes the competitive inhibition.
Molecular Mass Determination of Chitinase
One method which is very efficient in separation
and resolution of protein components in the sample is
SDS-PAGE. Results of SDS-PAGE analysis from the

Indo. J. Chem., 2010, 10 (2), 263 - 267

chitinase crude of Bacillus sp. HSA,3-1a showed that
there were 5 protein bands observed with the estimated
molecular masses of 79, 71, 48, 43 and 22 kDa. The
protein sizes of chitinase are almost the same as that of
other chitinases produced from different isolates. The
molecular mass of chitinase from Bacillus MH-1 was 71,
62, and 53 kDa [5]. Two chitinase with different
molecular mass (30 and 60 kDa) are isolated from
P.aeuroginosa K.187 [4]. The one of chitinase from
Bacillus sp. K-29, was 67 kDa [9]. The result showed
that the molecular masses of protein/enzyme varied
based on their source.
CONCLUSION
Based on the research results, it can be concluded
that the optimum condition of chitinase production from
thermophilic bacteria, Bacillus sp. HSA,3-1a was as
o
follows: temperature of 55 C; medium pH of 7,0; colloidal
chitin concentration of 0.5%; fermentation time of 72 h
with the chitinase activity of 0.225 Unit/mL and the
protein content of 0.154 mg/ mL.
Chitinase crude from Bacillus sp. HSA,3-1a worked
optimally at the temperature of 60 °C, the phosphate
buffer pH of 7,0 and the substrate concentration of 0.3%.
2+
2+
The enzyme was activated by 1 mM Mg , Ca and
2+
2+
2+
2+
Mn ions and inhibited by 1 mM Co , Zn and Cu
ions. The estimated of molecular masses of the five
proteins obtained from the SDS-PAGE analysis were 79,
71, 48, 43 and 22 kDa.
ACKNOWLEDGEMENT
The authors wish to extend their sincere thanks to
the Directorate General of Higher Education, National
Education Ministry for providing fund to support the
research.

Hasnah Natsir et al.

267

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